Neoplastic cells often differ from normal cells in that they express several proteins abnormally. Due to this anomalous expression, some proteins can act as Tumor Associate Antigens (TAA). This is because the host immune system can recognize these abnormalities and elicit an immune response that might protect the host from tumor onset and development. To be a target of antitumoral immunotherapy, a TAA must:                have a pathogenetic role in a certain stage of neoplastic growth;        be detectable by immune system even when the tumor gives rise to clonal variants which no more express major histocompatibility complex (HLA) glicoproteins;        be recognized by both antibodies and T lymphocytes.        
Several TAA have been discovered in human carcinomas in recent years. Among them, p185neu, the protein product of Her-2/neu (ErbB2) oncogene, is a particularly suited target for immunotherapy (Lollini and Forni, 2003, Trends Immunol. 24: 62). p185neu is a membrane receptor of class I receptor tyrosin kinase family, which also encompasses the epidermal growth factor receptor (EGF-R or ErbB-1) and other related receptors (ErbB-3, ErbB-4) which play a key role in cell proliferation and differentiation (Hynes and Stern, 1994, BBA 1198: 165).
p185neu receptor protein can be subdivided into three domains: the extracellular domain (EC domain), the transmembrane domain (TM domain), and the intracytoplasmic domain (IC domain). Recently, the EC domain crystallographic structure of human and rat p185neu protein has been published. This domain has been described to be composed of four subdomains (I/L1, II/CR1, III/L2, and IV/CR2) for approximately 630 amino acids in all. It has been further shown that p185neu protein has a rigid conformation, which allows it to interact with other ErbB receptors, dimerize, and induce transduction of proliferation signal even if this protein binds no ligands directly (Cho et al., 2003, Nature 421: 756).
Her-2/neu (ErbB2) oncogene is involved in normal processes of embryonic organogenesis and epithelial growth, while in adults it is expressed only at faint levels (Press et al., 1990, Oncogene 5: 953). In humans, overexpression of this oncogene is mainly caused by gene amplification. Her-2/neu (ErbB2) oncogene is overexpressed in about 30% of mammary carcinomas, and such an overexpression is related to a more rapid tumor progression (Slamon et al., 1989, Science 244: 707). Among the different strategies which have been proposed, DNA vaccination seems to be an effective method to elicit an immune response to Her-2/neu-positive tumors. Even though p185neu protein is a “self” antigen, i.e. a protein which is normally present in the body, patients with p185neu-positive mammary carcinomas often exhibit an immune response, both cellular and humoral (Signoretti et al., 2000, J. Natl. Cancer Inst. 23: 1918; Disis et al., 1994, Cancer Res. 54: 16; Peoples et al., 1995, Proc. Natl. Acad. Sci. USA 92: 432). One of the objectives of antitumoral immunotherapies directed towards p185neu protein is to increase the response intensity in patients with a pre-existing immune response, or to generate an immune response in patients in whom this response is undetectable. The fact that p185neu protein is a “self” antigen entails that the vaccine must be able to overcome an immunotolerant state.
The inventors of the instant patent application were the first using and validating the efficacy of DNA vaccination in eliciting an immune protection both to spontaneous mammary carcinomas and transplantable Her-2/neu-positive tumors. These studies have proven that prevention and treatment of preneoplastic lesions is an accessible goal. In particular, in experiments aimed at preventing the development of spontaneous mammary tumors that arise in transgenic mice due to rat Her-2/neu oncogene (FVB/neuT mice and BALB-neuT mice), it has been shown that the plasmid coding for rat p185neu protein EC and TM domains is capable of eliciting a more effective protection compared to the plasmid coding for full-length p185neu protein or plasmid coding for its EC domain only (secreted antigen) (Amici et al., 2000, Gene Ther. 7: 703; Rovero et al., 2000, J. Immunol. 165: 5133). Similar data have been reported by Chen et al., (1998, Cancer Res. 58: 1965). Furthermore, it has been shown that efficacy of vaccination with DNA plasmids is strongly increased if it is followed by a very short electric pulse when plasmids are inoculated intramuscularly (Quaglino et al., 2004, Cancer Res. 64: 2858). Other authors have shown that plasmids coding for full-length p185neu protein, if necessary mutated such that it does not possess tyrosine kinase activity, are efficacious in preventing the onset of tumors following the transplant of p185neu-positive cancer cells (Wei-Zen et al., 1999, Int. J. Cancer 81: 748). The same plasmids have proven as much effective even when, deprived of the leader signal responsible for protein processing in the endoplasmic reticulum, they bring about the cytoplasmic localization of p185neu antigen. When plasmids coding for p185neu protein which localizes in membrane thanks to the presence of a leader signal are used, protections depends upon an immune response which relies on antibodies. On the contrary, a T lymphocyte-mediated immune response is observed if vaccine does not contain a leader signal, and hence p185neu protein localizes in the cytoplasm of transfected cells rather than on their plasma membrane (Pilon et al., 2001, J. Immunol. 167: 3201). In addition, a combined vaccination obtained by using both plasmids with a leader signal and those in which this leader signal has been deleted, is more effective in protecting against tumor growth (Piechocki et al., 2001, J. Immunol. 167: 3367). This demonstrates that there is a synergistic effect between humoral and cellular responses in the prevention of p185neu-positive carcinomas (Reilly et al., 2001, Cancer Res. 61: 880).
Vaccination with the plasmid coding for EC and TM domains (EC-TM plasmid) has proven efficacious not only in preventing the development of spontaneous p185neu-positive carcinomas, but also in treating tumor masses of 2 mm in diameter by involving a range of effector immune system mechanisms (T helper and T killer cells, antibodies, macrophages, neutrophils, natural killer cells, Fc receptors, IFN-gamma, and perforins), which coordinately contribute to tumor rejection (Curcio et al., 2003, J. Clin. Invest. 111: 1161).